Apoptosis of epitope-specific antiretroviral cytotoxic T lymphocytes via Fas ligand-Fas interactions.

C57BL/6 (B6; H-2b) mice are capable of mounting a vigorous AKR/Gross Murine Leukemia Virus (MuLV)-specific cytotoxic T lymphocyte (CTL) response to AKR/Gross MuLVs whereas AKR.H- 2b congenic mice, although carrying the responder H-2b major histocompatibility haplotype, are specifically nonresponsive. Furthermore, when viable AKR.H-2b spleen cells are cocultured with primed responder B6 antiviral precursor CTLs, the AKR.H-2b cells function as "veto" cells that actively mediate the inhibition by apoptosis of B6 antiviral CTL generation in a contact-dependent, MHC-restricted, and veto cell Fas ligand (FasL)/responder T cell Fas-dependent manner. In the present study we show that antigen-specific, antiviral CTLs that survive apoptotic inhibition by AKR.H-2b veto cells display a less activated cell surface phenotype, and are less able to bind specific MHC-peptide tetramers, including on a per-T cell receptor (TcR) basis. In addition, surviving antiviral CTLs also appeared to be functionally deficient, based on both their reduced ability to lyse specific target cells and to produce interferon (IFN)-gamma. Carboxyfluorescein diacetate succinimidyl ester staining confirmed that AKR/Gross MuLV-specific CTLs proliferated less extensively when AKR.H-2b veto cells were included in cocultures. AKR/Gross MuLV-specific effector CTLs as well as memory CTLs were each efficiently targeted for inhibition by AKR.H-2b veto cells. Attempts to enhance the quality of the priming by multiple in vivo immunizations did not alter the capacity of the AKR.H-2b cells to inhibit the antiviral CTL response. These results further characterize the nature of the interaction between veto cells and antiviral CTLs, and underscore the efficiency of veto cell-mediated inhibition of the CTL response.

Prenatal transmission and pathogenicity of endogenous ecotropic murine leukemia virus Akv.

OBJECTIVE: Mouse strains carrying endogenous ecotropic murine leukemia viruses (MuLV) are capable of expressing infective virus throughout life. Risk of transplacental transmission of MuLV raises concerns of embryo infection and induction of pathogenic effects, and postnatal MuLV infection may lead to tumorigenesis. METHODS: Endogenous ecotropic MuLV-negative SWR/J embryos were implanted into Akv-infected viremic SWR/J mice, into spontaneously provirus-expressing AKR/J mice, and into noninfected SWR/J control mice; virus integration and virus expression were investigated at 14 days' gestation. Tumor development was monitored over 18 months. RESULTS: Of 111 embryos, 20 (18%) recovered from Akv-infected SWR/J mice, which had developed normally, were infected. New proviruses were detected in 10 of 111 (9%) embryos from Akv-infected SWR/J mice, and in 2 of 60 (3%) embryos from AKR/J mice; none expressed viral protein. Of 127 embryos recovered from Akv-infected SWR/J mice, 16 (13%) were dead; 4 of 5 (80%) were infected and expressed viral protein. Of 71 embryos from AKR/J mice, 11 (15%) were dead, and 2 of 2 had virus integration; virus expression was not detected. Numbers of dead embryos recovered from experimentally infected, viremic SWR/J mice and from spontaneously endogenous MuLV-expressing AKR/J mice were significantly higher, compared with numbers from nonviremic SWR/J control mice, and embryo lethality was significantly associated with prenatal provirus expression. Postnatal inoculation of Akv induced lymphoblastic lymphomas in 15 of 24 (61%) SWR/J mice within mean +/- SD latency of 14 +/- 2.4 months. Only 3 of 39 (8%) control mice developed lymphomas (P < 0.005). CONCLUSION: Embryos in MuLV-viremic dams are readily infected, and inappropriate prenatal expression of leukemogenic endogenous retroviruses may play a critical role in embryo lethality and decreased breeding performance in ecotropic provirus-positive mouse strains.

Akv murine leukemia virus enhances bone tumorigenesis in hMT-c-fos-LTR transgenic mice.

hMt-c-fos-LTR transgenic mice (U. Rüther, D. Komitowski, F. R. Schubert, and E. F. Wagner. Oncogene 4, 861-865, 1989) developed bone sarcomas in 20% (3/15) of females at 448 +/- 25 days and in 8% (1/12) of males at 523 days. After infection of newborns with Akv, an infectious retrovirus derived from the ecotropic provirus of the AKR mouse, 69% (20/28) of female animals and 83% (24/29) of males developed malignant fibrous-osseous tumors. The tumors in infected transgenics developed with higher frequency and a 200-days shorter mean tumor latency period. The hMt-c-fos-LTR transgene was expressed in all the fibrous-osseous tumors. They also showed newly integrated Akv proviruses, but in most tumors Akv was detected and expressed in only a small number of the tumor cells. Wild-type C3H mice infected with Akv developed benign osteomas with an incidence of 33% and a latency period of 474 days. The data indicate that Akv exerts distinct pathogenic effects on the skeleton. In hMt-c-fos-LTR transgenic mice, predisposed to bone sarcomagenesis, Akv acts synergistically with the fos transgene, resulting in the development of fibrous-osseous tumors.

Akv murine leukemia virus enhances lymphomagenesis in myc-kappa transgenic and in wild-type mice.

The contribution of endogenous retroviruses to the multistep process of lymphomagenesis was investigated in wild-type mice and in two different myc-kappa transgenic mouse lines by infection with Akv. This retrovirus is derived from the endogenous ecotropic provirus of the AKR mouse and was previously considered to be nonlymphomagenic. The mice of the two myc-k transgenic lines are predisposed to B-cell lymphomagenesis and were therefore considered to be more susceptible to Akv. For comparison, the same mouse strains were also infected with the exogenous Moloney murine leukemia virus (MoMuLV). Both MoMuLV and Akv increased the tumor incidence and shortened the tumor latency period in wild-type mice and in the transgenic mouse lines. The differences in pathogenicity, number of provirus integrations, and level of virus expression between MoMuLV and Akv indicate different mechanisms of lymphomagenesis: while MoMuLV induced tumors apparently by insertional mutagenesis involving common integration sites similar to previous reports, the enhancement of lymphomagenesis by Akv seems to be directed by other mechanisms.

Specific infection of central nervous system white matter by a variant of gross murine leukemia virus.

Exposure of neonatal Balb.B mice to a variant of Gross murine leukemia virus, termed WB91-GV, resulted in selective white matter infection within the central nervous system. Viral antigens were detected in brain sections of animals inoculated by either intracerebral or intraperitoneal routes, but were only seen in mice exposed within the first day after birth. This distinct tropism was confirmed by virus replication and gp70 expression in isolated glial cultures in vitro. Analysis of gp70 expression in highly enriched glial subpopulations indicated that oligodendrocytes and perhaps a subset of astrocytes were the targets of this infection.

Phenotypic heterogeneity of Gross virus-induced thymic lymphomas in the rat: cellular origins and migratory properties.

Neoplastic thymocytes from rat thymic lymphoma-leukemias induced by the rat-adapted Gross leukemia virus (RAGV) were analyzed for a variety of differentiation markers. The neoplasms from individual rats all expressed the antigenic phenotype MP+, W3/13+, Thy-1+, RT-1+, RT-7+, W3/25-. However, approximately two-thirds of the neoplasms were positive for the OX 8 antigen, and one-third were negative. The OX 8- neoplasms only involved the thymus, whereas approximately 40% of the OX 8+ neoplasms involved the spleen as well as the thymus. Virtually all OX 8+ and OX 8- neoplastic cells contained terminal deoxynucleotidyl transferase (TdT), and both OX 8+ and OX 8- lymphomas expressed the lactate dehydrogenase (LDH)-5' isozyme and the primary, but not the secondary, ADA isozyme. This enzymatic phenotype is characteristic of thymocyte precursors, but not thymocytes. Our results therefore indicate that RAGV-induced lymphomas arise from transformed prethymic TdT+ cells which contain the LDH-5' and the primary ADA isozymes. These preleukemic cells presumably migrate to the thymus where they express the RT-7 pan-T-cell antigen and, in some instances, the OX 8 antigen during the development of overt leukemia. The OX 8+ neoplasms, being more differentiated than their OX 8- counterparts, then migrate to peripheral lymphoid tissues.

The tandem direct repeats within the long terminal repeat of murine leukemia viruses are the primary determinant of their leukemogenic potential.

To map the viral sequences encoding the leukemogenic determinant(s) of nondefective murine leukemia viruses (MuLVs), we constructed chimeric viral genomes in vitro between cloned viral DNAs from the highly leukemogenic Gross passage A (Gross A) MuLV and from the related nonleukemogenic BALB/c N-tropic MuLV. Infectious chimeric MuLVs, recovered from murine cells microinjected with these DNAs, were inoculated into newborn mice to test the leukemogenic potential of these viruses. We found that the U3 long terminal repeat region from Gross A genomes was sufficient to confer an intermediate leukemogenic potential to chimeric MuLVs. Sequencing data indicated that the U3 tandem direct repeat was responsible for this effect. Adding most of the Gross A p15E-coding sequences to the Gross A U3 long terminal repeat enhanced the leukemogenic potential of chimeric viruses significantly. Adding a larger 3'-end env region (all p15E-coding sequences and 345 base pairs of the carboxy terminus of gp70) to the Gross A U3 long terminal repeat restored the full leukemogenic potential of Gross A MuLV. Chimeric viruses harboring only the Gross A 3'-end env region were, however, nonleukemogenic. Similar chimeric MuLVs, constructed with genomes from the parental weakly leukemogenic BALB/c B-tropic MuLVs and nonleukemogenic BALB/c N-tropic MuLVs, were also studied. Our data indicate that the U3 tandem direct repeat sequences appear to be necessary and sufficient to confer some leukemogenic potential to MuLV. However, env 3'-end sequences, mostly the p15E-encoding sequences, are required for the expression of fully leukemic phenotypes.

Low incidence of gross leukemia virus-induced lymphomas in spontaneously hypertensive rats with thymic dysfunction.

We compared the incidence of lymphomas induced by Gross leukemia virus (GLV) between spontaneously hypertensive rats (SHR) with a congenital T-cell depression related to thymic dysfunction and normal Wistar rats, the original strain of SHR. Of 20 SHR given neonatal injections of GLV, only 3 (15%) died with thymic lymphomas about 100 days after the virus infection. In contrast, 27 of 28 Wistar rats (96%) developed lymphomas of mostly thymic origin. The 3 lymphomas derived from the SHR bore only a Thy 1.1 antigen, whereas most of the lymphomas derived from Wistar rats carried not only a Thy 1.1 antigen but also a guinea pig red blood cell rosette receptor and a T (W3/13) antigen. Grafts of 1-week-old male Wistar thymus into the neonatal female SHR promoted a differentiation of thymocytes and markedly increased the incidence of the lymphomas which were positive for a guinea pig red blood cell rosette receptor and a T-antigen; grafts of 1-week-old SHR thymus, however, failed to do this. These results suggest that the low incidence of GLV-induced lymphomas in SHR may correlate closely with the absence or decreased numbers of the rosette-forming thymocytes which are presumably the target cells for GLV.

The high leukemogenic potential of Gross passage A murine leukemia virus maps in the region of the genome corresponding to the long terminal repeat and to the 3' end of env.

The Gross passage A murine leukemia virus (MuLV) is a highly leukemogenic, ecotropic fibrotropic retrovirus. Its genome is similar to that of other nonleukemogenic ecotropic fibrotropic MuLVs but differs at the 3' end and in the long terminal repeat. To determine whether these modifications were related to its leukemogenic potential, we constructed a viral DNA recombinant in vitro with cloned infectious DNA from this highly leukemogenic Gross passage A MuLV and from a weakly leukemogenic endogenous BALB/c B-tropic MuLV. Infectious viruses, recovered after microinjection of murine cells with recombinant DNA, were injected into newborn mice. We show here that the Gross passage A 1.35-kilobase-pair KpnI fragment (harboring part of gp70, all of p15E, and the long terminal repeat) is sufficient to confer a high leukemogenic potential to this recombinant.

Leukemogenesis by Gross passage A murine leukemia virus: expression of viruses with recombinant env genes in transformed cells.

Gross passage A murine leukemia virus (MuLV) derived from extracts of C3Hf/Bi mouse leukemias has been shown to be a virus complex consisting of ecotropic, xenotropic, and recombinant, dualtropic MuLV components. The three virus components were distinguished biochemically by differences in the molecular weights and peptide maps of their primary env gene products synthesized in infected cells in vivo and in vitro. Virus expression was studied in primary leukemias induced in C3Hf/Bi mice by Gross passage A virus extracts and by the individual ecotropic and recombinant MuLV components that were isolated in vitro. Our findings suggest that expression of the recombinant MuLV component of the Gross passage A virus complex is necessary and sufficient for the induction of leukemias in C3Hf/Bi mice. In contrast, induction of leukemias by the ecotropic virus component appears to involve generation of a second virus with characteristics of recombinant, dualtropic MuLV.

Most sequence differences between the genomes of the Akv virus and a leukemogenic Gross A virus passaged in vitro are located near the 3' terminus.

The 70S genomic RNA of nonleukemogenic AKR(Akv) virus was compared to that of an in vitro passaged, cloned, leukemogenic Gross A virus by fingerprint and sequence analysis. Fifty-seven of the large ribonuclease T1-resistant oligonucleotides of each virus have the same electrophoretic mobility and sequence. Thirteen large ribonuclease T1 oligo nucleotides are unique to the Gross A virus, whereas five are unique to Akv. Four of the oligonucleotides unique to each virus are related by one or two simple base changes. Five of the differences in oligonucleotides are located in the region of the genome that codes for the gag and pol genes. Eight of the differences are located near the 3' poly(A) terminus of the virus. The origins and biological consequences of these differences are discussed.

Relative loss of oncogenic potency of mouse leukemia virus (Gross) after prolonged propagation in tissue culture.

Since the initial development of the "passage A" mouse leukemia virus in 1957, this virus has been propagated in our laboratory by serial passage in newborn C3H(f) mice. At the present time, 10(-2)-10(-3) dilutions in physiological saline solution of this mouse-passaged virus induce lymphatic leukemia in practically all inoculated mice after a latency of 3-5 months. On the other hand, when the same virus was propagated on NIH 3T3 mouse embryo cells in tissue culture for more than 10 years, its leukemogenic potency became considerably reduced. Recent bioassay experiments carried out in our laboratory demonstrated that after such prolonged propagation in tissue culture this virus now induced leukemia in less than 15% of the inoculated suckling C3H(f) mice; only undiluted or 10% dilutions of the tissue culture fluid (very occasionally 10(-2) or 10(-3) dilutions) induced leukemia after a prolonged latency varying from 5.5 to 18 months. The passaged and the tissue-culture-grown virus strains are identical immunologically and indistinguishable in their morphology when examined by electron microscopy. The tissue-culture-grown virus, attenuated in its leukemogenic potency, does not, however, confer immunity against a challenge with the mouse-passaged virus.

Oncogenicity of AKR endogenous leukemia viruses.

Four biologically distinct groups of endogenous murine leukemia virus (MuLV) have been isolated from AKR mice. These viruses included (i) ecotopic XC+ MuLV that occur in high titer in normal tissues and serum of AKR mice throughout their life span, (ii) ecotropic XC- MuLV that are produced in high titers by leukemia cells, (iii) xenotropic MuLV that are readily demonstrable only in aged mice, and (iv) polytropic MuLV thatarise in the thymuses of aged mice as a consequence of genetic recombination between ecotropic and xenotropic MuLV. Virus of each of these biological classes were assayed in AKR mice for their ability to accelerate the occurrence of spontaneous leukemia. Certain isolates of ecotropic XC- MuLV and polytropic MuLV were found to have high oncogenic activity. These viruses induced 100% leukemias within 90 days of inoculation. In contrast, ecotropic XC+ MuLV that were obtained from AKR embryo fibroblasts and xenotropic MuLV that were obtained from the lymphoid tissues of aged AKR mice did not demonstrate oncogenic activity. These findings demonstrate fundamental differences between XC- and XC+ ecotropic MuLV that are found in leukemic and normal tissues, respectively. Furthermore, these findings point to the role of ecotropic XC- and polytropic MuLV in the spontaneous leukemogenesis of AKR mice.